Retinoic Acid Receptor Alpha Research Articles

Vitamin A deficiency and vitamin A supplementation affect innate and T cell immune responses to rotavirus A infection in a conventional sow model.

Rotavirus A (RVA) causes ~200,000 diarrheal deaths annually in children <5yrs, mostly in low- and middle-income countries. Risk factors include nutritional status, social factors, breastfeeding status, and immunodeficiency. We evaluated the effects of vitamin A (VA) deficiency/VA supplementation and RVA exposure (anamnestic) on innate and T cell immune responses in RVA seropositive pregnant and lactating sows and passive protection of their piglets post-RVA challenge. Sows were fed VA deficient (VAD) or sufficient (VAS) diets starting at gestation day (GD)30. A subset of VAD sows received VA supplementation from GD|76 (30,000IU/day, VAD+VA). Sows (6 groups) were inoculated with porcine RVA G5P[7] (OSU strain) or Minimal Essential Medium (mock) at GD~90: VAD+RVA; VAS+RVA; VAD+VA+RVA; VAD-mock; VAS-mock; and VAD+VA-mock. Blood, milk, and gut-associated tissues were collected from sows at several time points to examine innate [natural killer (NK), dendritic (DC) cells], T cell responses and changes in genes involved in the gut-mammary gland (MG)-immunological axis trafficking. Clinical signs of RVA were evaluated post inoculation of sows and post-challenge of piglets. We observed decreased frequencies of NK cells, total and MHCII+ plasmacytoid DCs, conventional DCs, CD103+ DCs and CD4+/CD8+ and T regulatory cells (Tregs) and NK cell activity in VAD+RVA sows. Polymeric Ig receptor and retinoic acid receptor alpha (RARα) genes were downregulated in mesenteric lymph nodes and ileum of VAD+RVA sows. Interestingly, RVA-specific IFN-γ producing CD4+/CD8+ T cells were increased in VAD-Mock sows, coinciding with increased IL-22 suggesting inflammation in these sows. VA supplementation to VAD+RVA sows restored frequencies of NK cells and pDCs, and NK activity, but not tissue cDCs and blood Tregs. In conclusion, similar to our recent observations of decreased B cell responses in VAD sows that led to decreased passive immune protection of their piglets, VAD impaired innate and T cell responses in sows, while VA supplementation to VAD sows restored some, but not all responses. Our data reiterate the importance of maintaining adequate VA levels and RVA immunization in pregnant and lactating mothers to achieve optimal immune responses, efficient function of the gut-MG-immune cell-axis and to improve passive protection of their piglets.

De novo variants implicate chromatin modification, transcriptional regulation, and retinoic acid signaling in syndromic craniosynostosis

Craniosynostosis (CS) is the most common congenital cranial anomaly. Several Mendelian forms of syndromic CS are well described, but a genetic etiology remains elusive in a substantial fraction of probands. Analysis of exome sequence data from 526 proband-parent trios with syndromic CS identified a marked excess (observed 98, expected 33, p= 4.83×10-20) of damaging de novo variants (DNVs) in genes highly intolerant to loss-of-function variation (probability of LoF intolerance>0.9). 30 probands harbored damaging DNVs in 21 genes that were not previously implicated in CS but are involved in chromatin modification and remodeling (4.7-fold enrichment, p= 1.1×10-11). 17 genes had multiple damaging DNVs, and 13 genes (CDK13, NFIX, ADNP, KMT5B, SON, ARID1B, CASK, CHD7, MED13L, PSMD12, POLR2A, CHD3, and SETBP1) surpassed thresholds for genome-wide significance. A recurrent gain-of-function DNV in the retinoic acid receptor alpha (RARA; c.865G>A [p.Gly289Arg]) was identified in two probands with similar CS phenotypes. CS risk genes overlap with those identified for autism and other neurodevelopmental disorders, are highly expressed in cranial neural crest cells, and converge in networks that regulate chromatin modification, gene transcription, and osteoblast differentiation. Our results identify several CS loci and have major implications for genetic testing and counseling.

Treating low- and intermediate-risk acute promyelocytic leukemia with and without chemotherapy: A comparison in a tertiary care center.

Acute promyelocytic leukemia (APL) comprises approximately 10% of acute myeloid leukemia (AML) cases. Both options of treatment (ATRA-ATO and ATRA-chemotherapy) were discussed with patients with low- and intermediate-risk APL, pros and cons explained in details, and treatment regimen selected after getting informed written consent. Total 71 patients were included in the study; among these patients, 3 were negative for both FISH for t (15,17) and RT-PCR for promyelocytic leukemia retinoic acid receptor alpha, and 36 patients with APL had white blood cell count at diagnosis >10 × 109/l. Total 30 patients with newly diagnosed as low- and intermediate-risk-APL fulfilled all inclusion criteria, treated and followed for a minimum period of 2 years up to June, 2016. Fifteen patients liked to be treated with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), while rest of the 15 patients preferred treatment with ATRA and chemotherapy. Combination of ATRA and ATO is equally effective, less toxic, and more feasible in comparison to ATRA and chemotherapy for patients with low- and intermediate-risk APL and is a viable option for this subset of patients, especially in countries with limited resources.

Molecular Heterogeneity of Pediatric AML with Atypical Promyelocytes Accumulation in Children-A Single Center Experience.

Acute promyelocytic leukemia (APL) pathogenesis is based on RARA gene translocations, which are of high importance in the diagnosis of and proper therapy selection for APL. However, in some cases acute myeloid leukemia (AML) demonstrates APL-like morphological features such as atypical promyelocytes accumulation. This type of AML is characterized by the involvement of other RAR family members or completely different genes. In the present study, we used conventional karyotyping, FISH and high-throughput sequencing in a group of 271 de novo AML with atypical promyelocytes accumulation. Of those, 255 cases were shown to carry a typical chromosomal translocation t(15;17)(q24;q21) with PML::RARA chimeric gene formation (94.1%). Other RARA-positive cases exhibited cryptic PML::RARA fusion without t(15;17)(q24;q21) (1.8%, n = 5) and variant t(5;17)(q35;q21) translocation with NPM1::RARA chimeric gene formation (1.5%, n = 4). However, 7 RARA-negative AMLs with atypical promyelocytes accumulation were also discovered. These cases exhibited TBL1XR1::RARB and KMT2A::SEPT6 fusions as well as mutations, e.g., NPM1 insertion and non-recurrent chromosomal aberrations. Our findings demonstrate the genetic diversity of AML with APL-like morphological features, which is of high importance for successful therapy implementation.

The p97/VCP segregase is essential for arsenic-induced degradation of PML and PML-RARA.

Acute Promyelocytic Leukemia is caused by expression of the oncogenic Promyelocytic Leukemia (PML)-Retinoic Acid Receptor Alpha (RARA) fusion protein. Therapy with arsenic trioxide results in degradation of PML-RARA and PML and cures the disease. Modification of PML and PML-RARA with SUMO and ubiquitin precedes ubiquitin-mediated proteolysis. To identify additional components of this pathway, we performed proteomics on PML bodies. This revealed that association of p97/VCP segregase with PML bodies is increased after arsenic treatment. Pharmacological inhibition of p97 altered the number, morphology, and size of PML bodies, accumulated SUMO and ubiquitin modified PML and blocked arsenic-induced degradation of PML-RARA and PML. p97 localized to PML bodies in response to arsenic, and siRNA-mediated depletion showed that p97 cofactors UFD1 and NPLOC4 were critical for PML degradation. Thus, the UFD1-NPLOC4-p97 segregase complex is required to extract poly-ubiquitinated, poly-SUMOylated PML from PML bodies, prior to degradation by the proteasome.

Poly(ethylene glycol) diacrylate based hydrogel filled micropore with enhanced sensing capability

Ionic current rectification (ICR) phenomena conventionally occurs in nanopores which dimensions are comparable to the thickness of their electrical double layers. However, the microscale ICR in a micropore can also exist under some conditions. Here, the charged hydrogel filled conical micropore was constructed to realize microscale ICR. To better understand the micropore ICR, the influences of space charge density of the hydrogel, micropore geometry, the hydrogel filling length as well as the electrolyte concentration and pH were investigated. Furthermore, we developed a PEGDA-based hydrogel filled micropore sensing platform which sensing performance was enhanced due to the weakly charged PEGDA. The promyelocytic leukemia (PML)/retinoic acid receptor alpha (RARA) fusion genes and adenosine triphosphate (ATP) were respectively used as model analytes and the measured detection limits of 0.1 pM were achieved. The successful realization of microscale ICR in a homogenous and functional hydrogel filled micropore suggests that the fabrication, characterization and operation of ICR based devices can be more robust and facilitated for the wider applications.

Activation of the RARα Attenuated CSF Hypersecretion to Inhibit Hydrocephalus Development via Regulating the MAFB/MSR1 Pathway.

Hydrocephalus has been observed in rats with spontaneous hypertension (SHRs). It has been demonstrated that activation of the oxidative stress related protein retinoic acid receptor alpha (RARα) has neuroprotective impacts. Our investigation aims to determine the potential role and mechanism of RARα in hydrocephalus. The RARα-specific agonist (Am80) and RARα inhibitor (AGN196996) were used to investigate the role of RARα in cerebrospinal fluid (CSF) secretion in the choroid plexus of SHRs. Evaluations of CSF secretion, ventricular volume, Western blotting, and immunofluorescent staining were performed. Hydrocephalus and CSF hypersecretion were identified in SHRs but not in Wistar-Kyoto rats, occurring at the age of 7 weeks. The RARα/MAFB/MSR1 pathway was also activated in SHRs. Therapy with Am80 beginning in week 5 decreased CSF hypersecretion, hydrocephalus development, and pathological changes in choroid plexus alterations by week 7. AGN196996 abolished the effect of Am80. In conclusion, activation of the RARα attenuated CSF hypersecretion to inhibit hydrocephalus development via regulating the MAFB/MSR1 pathway. RARα may act as a possible therapeutic target for hydrocephalus.

The Role of Apoptotic Genes and Protein-Protein Interactions in Triple-negative Breast Cancer.

Compared to other breast cancer types, triple-negative breast cancer (TNBC) has historically had few treatment alternatives. Therefore, exploring and pinpointing potentially implicated genes could be used for treating and managing TNBC. By doing this, we will provide essential data to comprehend how the genes are involved in the apoptotic pathways of the cancer cells to identify potential therapeutic targets. Analysis of a single genetic alteration may not reveal the pathogenicity driving TNBC due to the high genomic complexity and heterogeneity of TNBC. Therefore, searching through a large variety of gene interactions enabled the identification of molecular therapeutic genes. This study used integrated bioinformatics methods such as UALCAN, TNM plotter, PANTHER, GO-KEEG and PPIs to assess the gene expression, protein-protein interaction (PPI), and transcription factor interaction of apoptosis-regulated genes. Compared to normal breast tissue, gene expressions of BNIP3, TNFRSF10B, MCL1, and CASP4 were downregulated in UALCAN. At the same time, BIK, AKT1, BAD, FADD, DIABLO, and CASP9 was down-regulated in bc-GeneExMiner v4.5 mRNA expression (BCGM) databases. Based on GO term enrichment analysis, the cellular process (GO:0009987), which has about 21 apoptosis-regulated genes, is the top category in the biological processes (BP), followed by biological regulation (GO:0065007). We identified 29 differentially regulated pathways, including the p53 pathway, angiogenesis, apoptosis signaling pathway, and the Alzheimer's disease presenilin pathway. We examined the PPIs between the genes that regulate apoptosis; CASP3 and CASP9 interact with FADD, MCL1, TNF, TNFRSRF10A, and TNFRSF10; additionally, CASP3 significantly forms PPIs with CASP9, DFFA, and TP53, and CASP9 with DIABLO. In the top 10 transcription factors, the androgen receptor (AR) interacts with five apoptosis-regulated genes (p<0.0001; q<0.01), followed by retinoic acid receptor alpha (RARA) (p<0.0001; q<0.01) and ring finger protein (RNF2) (p<0.0001; q<0.01). Overall, the gene expression profile, PPIs, and the apoptosis-TF interaction findings suggest that the 27 apoptosis-regulated genes might be used as promising targets in treating and managing TNBC. Furthermore, from a total of 27 key genes, CASP2, CASP3, DAPK1, TNF, TRAF2, and TRAF3 were significantly correlated with poor overall survival in TNBC (p-value <0.05); they could play important roles in the progression of TNBC and provide attractive therapeutic targets that may offer new candidate molecules for targeted therapy. Our findings demonstrate that CASP2, CASP3, DAPK1, TNF, TRAF2, and TRAF3 were substantially associated with the overall survival rate (OS) difference of TNBC patients out of a total of 27 specific genes used in this study, which may play crucial roles in the development of TNBC and offer promising therapeutic interventions.

Initial Results from SELECT-AML-1, a Phase 2 Study of Tamibarotene in Combination with Venetoclax and Azacitidine in RARA-Positive Newly Diagnosed AML Patients Ineligible for Standard Induction Chemotherapy

RARA-positive AML is a novel genomically-defined subset with an actionable target for treatment with tamibarotene, an oral and selective RARα agonist (McKeown, Cancer Discovery 2017). Approximately 30% of non-APL AML patients are RARA-positive based on overexpression of the RARA gene in peripheral blood blasts as determined by an RT-PCR based assay (de Botton, ESH 2019). In patients with newly diagnosed (ND) AML ineligible for standard induction chemotherapy, tamibarotene/azacitidine led to a CR/CRi rate of 61% in RARA-positive patients with a rapid onset of response. The combination was well-tolerated with no increase in myelosuppression compared to azacitidine (aza) alone (de Botton, ASH 2020). Responses were observed irrespective of mutations or cytogenetic risk. Approximately one-third of ND AML patients ineligible for standard induction therapy do not respond to venetoclax (ven) and nearly all eventually relapse (DiNardo, NEJM 2020). Translational data suggest that RARA-positive patients are enriched for monocytic features based on a positive Monocytic Expression Score (MES) that may be associated with ven resistance (Fiore, ASH 2020), suggesting that the blood-based RARA biomarker test selects for patients who may respond to tamibarotene and who may also be less likely to respond to ven/aza alone. SELECT-AML-1 (NCT04905407) is a Phase 2, open-label, multi-center study in the US and France comparing the activity of tamibarotene/ven/aza to ven/aza in treatment-naive RARA-positive ND AML patients ineligible for standard induction therapy. The 3-part study includes a safety lead-in of the tamibarotene/ven/aza combination, a randomized efficacy study versus ven/aza, and a salvage arm for ven/aza only patients with primary endpoints of safety, CR/CRi rate, and overall response rate, respectively. Following the safety lead-in, patients will be randomized 1:1 to receive tamibarotene/ven/aza or ven/aza. In the salvage arm, patients randomized to ven/aza only who experience progressive disease, treatment failure, or relapse, will have tamibarotene added to their ven/aza regimen. Patients currently enrolled in the safety lead-in are treated with starting doses of aza at 75 mg/m2 IV/SC daily on Days 1-7, ven on Days 1-28 per the ven USPI/SMPC, followed by tamibarotene at 6 mg twice per day PO on Days 8-28 of each 28-day cycle. Enrollment is ongoing. As of 22 June 2022, six patients have enrolled to date, including four who are response-evaluable. Baseline characteristics of the response-evaluable patients include 75% male; median age 57 (55-74); median bone marrow blasts 55% (39-100%); cytogenetically normal (n=2), inversion 16 (n=1), del 5q and -7 (n=1); and molecular genetic testing normal (n=3), mutations of IDH2, BCOR, SRSF2, CSF3R, RUNX1 (n=1). The MES was high in 3 of 4 (75%) response evaluable patients consistent with a prior report in RARA-positive AML (Fiore, ASH 2020). All 4 response-evaluable patients achieved a best response of CR/CRi (2 CR, 2 CRi). The onset of response was rapid with all patients achieving an initial response after Cycle 1 (2 CR, 1 CRi, 1 MLFS). Median duration of treatment was 68 days (21-110). Two patients discontinued, both in remission, one due to death from neutropenic sepsis and the other by physician decision. The adverse event (AE) profile of the triplet was consistent with reports of ven/aza in AML. AEs experienced by 2 or more patients (all grades/causality) included febrile neutropenia, pneumonia, anxiety, cough, diarrhea, fatigue, decreased appetite, myalgia, decreased neutrophil count, and decreased platelet count. The only serious AE (all causality) occurring in more than one patient was febrile neutropenia. A high response rate was observed in early results of tamibarotene/ven/aza in RARA-positive ND AML patients ineligible for standard induction chemotherapy with 100% (4 of 4) response evaluable patients achieving CR/CRi, 3 of whom may have features of ven-resistance based on high MES. The triplet demonstrated an initial safety profile similar to ven/aza. Data from the safety lead-in will inform triplet dosing for the randomized portion of the study.

How retinoic acid and arsenic transformed acute promyelocytic leukemia therapy.

Acute promyelocytic leukemia (APL) is associated with severe coagulopathy leading to rapid morbidity and mortality if left untreated. The definitive diagnosis of APL is made by identifying a balanced reciprocal translocation between chromosomes 15 and 17. This t(15;17) results in a fusion transcript of promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARA) genes and the expression of a functional PML/RARA protein. Detection of a fused PML/RARA genomic DNA sequence using fluorescence in situ hybridization (FISH) or by detection of the PML/RARA fusion transcript via reverse transcriptase polymerase chain reaction (RT-PCR) has revolutionized the diagnosis and monitoring of APL. Once confirmed, APL is cured in over 90% of cases, making it the most curable subtype of acute leukemia today. Patients with low-risk APL are successfully treated using a chemotherapy-free combination of all-trans retinoic acid and arsenic trioxide (ATO). In this review, we explore the work that has gone into the modern-day diagnosis and highly successful treatment of this once devastating leukemia.

81. Discrepancies in the detection of PML::RARA gene rearrangement by FISH using commonly used dual-color dual-fusion probes

Acute promyelocytic leukemia (APL) is a medical emergency with serious complications (e.g., DIC), requires prompt treatment and rapid test turnaround time (TAT) for diagnosis confirmation. APL results from a fusion of the PML gene (15q24) with the RARA gene (17q21) creating a fusion gene on the derivative chromosome 15. There are multiple breakpoints that can occur. PML::RARA is the most common (98%) rearrangement. Fluorescent in situ Hybridization (FISH) study helps in the diagnosis of APL with rapid TAT. PML::RARA dcdf FISH probes from either Abbott Molecular or Cytocell are commonly used in the Cytogenetics laboratories. Although they both detect PML::RARA gene rearrangements they differ in their probe design, resulting in the limitation of breakpoint coverage in atypical PML-RARA insertion rearrangements. Our lab has identified two unique cases of APL which were tested at diagnosis using PML::RARA FISH probe set. Both cases showed an atypical rearrangement with single fusion signal. First case was positive by Abbott probe and negative by Cytocell, metaphase FISH confirmed insertion of PML region near RARA on Chromosome 17. Other case was positive by Cytocell and negative by Abbott, metaphase FISH confirmed insertion of RARA region near PML on chromosome 15. Both cases were positive for PML::RARA transcript presence by qRT-PCR. This data suggests potential limitation of widely used PML::RARA FISH probes from different FISH probe vendors. In cases with high suspicion of APL with a negative FISH result, reflex FISH test using probes having coverage different from initial tested probe and molecular PCR

Intracranial hypertension associated with treatment of acute promyelocytic leukemia with all-trans retinoic acid (ATRA): a case report

Case presentation&#x0D; Female, 18 years-old, without relevant family history, referred to Neurology by Hematology for headache. 3 years ago, she was diagnosed with Acute Promyelocytic Leukemia (APL) currently on maintenance therapy with All-Trans Retinoic Acid (ATRA). The patient reports that in all previous cycles presented headache with the following characters: one-side temporal location, pulsating quality, moderate intensity, with nausea and vomiting, photophobia and phonophobia, uncertain duration, with simple analgesics response. In the current cycle, she relates continuous pain, severe intensity and unresponsive to medication. On examination, there were no focal neurological findings and on funduscopic examination there was no papilledema. MRI and laboratorial tests were normal. Cerebrospinal fluid (CSF) opening pressure was 35 cmH2O, no other alterations. 15 milliliters of CSF were removed, with a closing pressure of 25 cmH2O. A hypothesis of Intracranial Hypertension associated with use of ATRA was made. Afterwards, there was important improvement of the headache, with residual pain of mild intensity. Therefore, Acetazolamide was started at a dose of 250 milligrams every 12 hours with complete resolution of symptoms.&#x0D; &#x0D; Discussion&#x0D; Acute myeloid leukemia (AML) comprises a heterogeneous group of aggressive blood cell cancers that arise from clonal expansion of malignant hematopoietic precursor cells in the bone marrow. Acute promyelocytic leukemia (APL) is a biologically and clinically distinct variant of AML. APL is classified as acute promyelocytic leukemia with PML-RARA. The cytogenetic hallmark of APL is a translocation involving RARA, the retinoic acid receptor alpha locus on chromosome 17. Without treatment, APL is the most malignant form of AML, with a median survival of less than one month.&#x0D; (To see the complete abstract, please, check out the PDF).

Retinoic Acid Receptor Alpha (RARα) in Macrophages Protects from Diet-Induced Atherosclerosis in Mice.

Retinoic acid signaling plays an important role in regulating lipid metabolism and inflammation. However, the role of retinoic acid receptor alpha (RARα) in atherosclerosis remains to be determined. In the current study, we investigated the role of macrophage RARα in the development of atherosclerosis. Macrophages isolated from myeloid-specific Rarα-/- (RarαMac-/-) mice showed increased lipid accumulation and inflammation and reduced cholesterol efflux compared to Rarαfl/fl (control) mice. All-trans retinoic acid (AtRA) induced ATP-binding cassette subfamily A member 1 (Abca1) and Abcg1 expression and cholesterol efflux in both RarαMac-/- mice and Rarαfl/fl mice. In Ldlr-/- mice, myeloid ablation of RARα significantly reduced macrophage Abca1 and Abcg1 expression and cholesterol efflux, induced inflammatory genes, and aggravated Western diet-induced atherosclerosis. Our data demonstrate that macrophage RARα protects against atherosclerosis, likely via inducing cholesterol efflux and inhibiting inflammation.

Effect of All-trans Retinoic Acid on Panniculus Carnosus Muscle Regeneration in Fetal Mouse Wound Healing.

Background:The dermal panniculus carnosus (PC) muscle is critical for wound contraction in lower mammals and is a useful model of muscle regeneration owing to its high cellular metabolic turnover. During wound healing in mice, skin structures, including PC, are completely regenerated up to embryonic day (E) 13, but PC is only partially regenerated in fetuses or adult animals after E14. Nevertheless, the mechanisms underlying wound repair for complete regeneration in PC have not been fully elucidated. We hypothesized that retinoic acid (RA) signaling, which is involved in muscle differentiation, regulates PC regeneration.Methods:Surgical injury was induced in ICR mice on E13 and E14. RA receptor alpha (RARα) expression in tissue samples from embryos was evaluated using immunohistochemistry and reverse transcription-quantitative polymerase chain reaction. To evaluate the effects of RA on PC regeneration, beads soaked in all-trans RA (ATRA) were implanted in E13 wounds, and tissues were observed. The effects of RA on myoblast migration were evaluated using a cell migration assay.Results:During wound healing, RARα expression was enhanced at the cut surface in PCs of E13 wounds but was attenuated at the cut edge of E14 PCs. Implantation of ATRA-containing beads inhibited PC regeneration on E13 in a concentration-dependent manner. Treatment of myoblasts with ATRA inhibited cell migration.Conclusions:ATRA inhibits PC regeneration, and decreased RARα expression in wounds after E14 inhibits myoblast migration. Our findings may contribute to the development of therapies to promote complete wound regeneration, even in the muscle.

Genetic Studies in Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) is a group of malignant diseases originating from clonal hematopoietic stem cells, with a prevalence of 2-3 per hundred thousand people all over the world every year, and whose incidence has not changed significantly for the last 20 years. This malignant change in hematopoietic cells causes loss of function in these cells and, if untreated, results in death within weeks or months depending on the clinical course. AML is a complex disease that shows heterogeneity as well as phenotypic and recurrent chromosomal aberrations are observed in most of the cases. AML-related translocations generally affect the CBF (corebinding factor), RARα (retinoic acid receptor alpha) and ETS (E-twentysix) family transcription factors and the HOX (Homeobox) gene family. There are various treatment examples and prognostic approaches as a result of genetic studies in AML. Recently, some molecules and molecular changes thought to be of prognostic and therapeutic importance have been identified and their importance in terms of treatment and prognosis has been investigated. In this study, genetic studies in AML were examined and new approaches and treatment processes in recent years were examined.

Targeting retinoic acid receptor alpha-corepressor interaction activates chaperone-mediated autophagy and protects against retinal degeneration

Chaperone-mediated autophagy activity, essential in the cellular defense against proteotoxicity, declines with age, and preventing this decline in experimental genetic models has proven beneficial. Here, we have identified the mechanism of action of selective chaperone-mediated autophagy activators previously developed by our group and have leveraged that information to generate orally bioavailable chaperone-mediated autophagy activators with favorable brain exposure. Chaperone-mediated autophagy activating molecules stabilize the interaction between retinoic acid receptor alpha - a known endogenous inhibitor of chaperone-mediated autophagy - and its co-repressor, nuclear receptor corepressor 1, resulting in changes of a discrete subset of the retinoic acid receptor alpha transcriptional program that leads to selective chaperone-mediated autophagy activation. Chaperone-mediated autophagy activators molecules activate this pathway in vivo and ameliorate retinal degeneration in a retinitis pigmentosa mouse model. Our findings reveal a mechanism for pharmacological targeting of chaperone-mediated autophagy activation and suggest a therapeutic strategy for retinal degeneration.

Expression and activation of nuclear hormone receptors result in neuronal differentiation and favorable prognosis in neuroblastoma

BackgroundNeuroblastoma (NB), a childhood tumor derived from the sympathetic nervous system, presents with heterogeneous clinical behavior. While some tumors regress spontaneously without medical intervention, others are resistant to therapy, associated with an aggressive phenotype. MYCN-amplification, frequently occurring in high-risk NB, is correlated with an undifferentiated phenotype and poor prognosis. Differentiation induction has been proposed as a therapeutic approach for high-risk NB. We have previously shown that MYCN maintains an undifferentiated state via regulation of the miR-17 ~ 92 microRNA cluster, repressing the nuclear hormone receptors (NHRs) estrogen receptor alpha (ERα) and the glucocorticoid receptor (GR).MethodsCell viability was determined by WST-1. Expression of differentiation markers was analyzed by Western blot, RT-qPCR, and immunofluorescence analysis. Metabolic phenotypes were studied using Agilent Extracellular Flux Analyzer, and accumulation of lipid droplets by Nile Red staining. Expression of angiogenesis, proliferation, and neuronal differentiation markers, and tumor sections were assessed by immunohistochemistry. Gene expression from NB patient as well as adrenal gland cohorts were analyzed using GraphPad Prism software (v.8) and GSEA (v4.0.3), while pseudo-time progression on post-natal adrenal gland cells from single-nuclei transcriptome data was computed using scVelo.ResultsHere, we show that simultaneous activation of GR and ERα potentiated induction of neuronal differentiation, reduced NB cell viability in vitro, and decreased tumor burden in vivo. This was accompanied by a metabolic reprogramming manifested by changes in the glycolytic and mitochondrial functions and in lipid droplet accumulation. Activation of the retinoic acid receptor alpha (RARα) with all-trans retinoic acid (ATRA) further enhanced the differentiated phenotype as well as the metabolic switch. Single-cell nuclei transcriptome analysis of human adrenal glands indicated a sequential expression of ERα, GR, and RARα during development from progenitor to differentiated chromaffin cells. Further, in silico analysis revealed that patients with higher combined expression of GR, ERα, and RARα mRNA levels had elevated expression of neuronal differentiation markers and a favorable outcome.ConclusionTogether, our findings suggest that combination therapy involving activation of several NHRs could be a promising pharmacological approach for differentiation treatment of NB patients.

Downregulation of hsa-miR-4328 and target gene prediction in Acute Promyelocytic Leukemia

Abstract Introduction: Acute promyelocytic leukemia (APL) is defined by the PML-RARA fusion gene. APL treatment can have significant side effects, therefore the development of optimal therapeutic options is crucial. Although the study of miRNAs is still in its infancy, it has been shown that these molecules are involved in the pathogenesis of neoplasms by modulating the expression of target genes. miRNAs can be considered possible biomarkers in APL and can be used as therapeutic targets or as markers for the therapeutic response. Objectives: The purpose of this study was to determine whether differentially expressed putative miRNAs that have RARA as a target gene could be considered reliable biomarkers for APL. Methods: Using bioinformatics tools, a panel of 6 miRNAs with possible tropism for the RARA gene was selected from miRDB. We evaluated their expression levels in samples from patients with APL (n=20) or from healthy subjects without mutations in genes associated with leukemia or myeloproliferative diseases (n=21). Results: All 6 putative miRNAs were identified using electrophoresis (hsamir-4299, hsa-mir-4328, hsa-mir-7851-3p, hsa-mir-6827-5p, hsa-mir-6867-5p, hsa-mir-939-5p). Of the six miRNAs, hsa-mir-4328 is deeply downregulated in subjects diagnosed with APL compared to healthy subjects, whereas hsa-mir-4299 and hsa-mir-7851-3p show small differences in expression between the two study groups, but without statistical significance. Our results suggest that hsa-mir-4328 may have a role in the pathogenesis of APL and may represent a new biomarker for this type of leukemia. Key Words: miRNA, APL, leukemia, bioinformatics.

Abstract 6067: Studying the anti-proliferating and differentiating effects of retinoids based on pathway genotype of colorectal cancer cell lines

Abstract Background: Our overarching goal is to therapeutically sensitize human colorectal cancer stem cells to differentiation-inducing effects of retinoid agents. Tumorigenesis in colorectal cancer (CRC) results from the acquisition of mutations in a colonic stem cell which transforms it into a cancer stem cell (CSC). There are no curative treatments for advanced CRC due to the resistance of CSCs to chemotherapy and/or radiation. All-trans retinoic acid (ATRA) is known to be a greatly effective treatment for acute promyelocytic leukemia (APL) which is caused by translocation in the retinoic acid receptor alpha (RARA) gene. We conjecture that the response of retinoic acid (RA) agents may have effects on solid tumors depending on their pathway genotype. Results from our bioinformatics analysis on mutations and overexpression of RA signaling component genes in CRC showed that the majority of RA pathway genes are overexpressed in CRC. Therefore, we hypothesize that the ability of retinoid and retinoid metabolism blocking agents to induce differentiating effects in CSCs depends on the RA pathway genotype expressed in CRC cells. Methods: The ability of RA agents (ATRA, 13-cis retinoic acid, 9-cis retinoic acid) and RA metabolism blocking agents (Liarozole and Talarozole) to inhibit proliferation and induce differentiation on CRC cell lines with different mutations in RA signaling were evaluated. Additionally, Nanostring Profiling was used to measure the effects of the agents on mRNA expression of CSC markers, indicators of cell differentiation, and RA receptors. Results: Our results show that the RA pathway genotype affects the response of RA agents in CRC cell lines. Both HCT116 and SW480 CRC cell lines, which are both mutant in RARA and RARA and RXRG respectively displayed resistance to ATRA. HT29 CRC cells express wild- type RA receptors and are sensitive to ATRA. All three cell lines showed similar dose responses to the CYP26A1 inhibitor Liarozole. This indicates that regardless of RA receptor mutations, inhibition of ATRA metabolism increases intracellular RA levels in a similar manner on all 3 cell lines. Nanostring Profiling shows that ATRA treatment of HT29 cells induces: an increase in the expression of RARA, CYP26A1 (ATRA metabolizing enzyme) and KRT20 (differentiation marker); and a decrease in the expression of CSC markers LGR5 and ALDH1A1. This indicates an active RA signaling pathway, induced differentiation, and a decrease in stem cells. Discussion: Overall, our findings indicate that colon SCs are regulated by RA signaling mechanisms and dysregulation of RA signaling contributes to the SC overpopulation that drives CRC growth. Therefore, further investigation on how to therapeutically sensitize human colonic CSCs to the differentiation-inducing effects of retinoid agents should provide insight into designing new SC-targeted therapies for CRC. Citation Format: Victoria O. Hunsu, Caroline O. Facey, Lynn M. Opdenaker, Bruce M. Boman. Studying the anti-proliferating and differentiating effects of retinoids based on pathway genotype of colorectal cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6067.

Deficiency of RARα Suppresses Decidualization via Downregulating CEBPB Transcription in Women With Recurrent Implantation Failure.

BackgroundRecurrent implantation failure (RIF) is a disease associated with endometrial receptivity dysfunction. Retinoic acid receptor alpha (RARα) is an important protein in many biological processes, such as differentiation and development. However, the exact underlying mechanism whereby RARα affects RIF remains unknown. This study investigated RARα expression and its contribution in the mid-luteal phase endometria of patients with RIF.MethodsThe expression levels of RARα and CCAAT/enhancer-binding protein (C/EBP) β in the endometria of the RIF and normal group were investigated using western blotting and immunohistochemistry. In in vitro experiments, immortal telomerase-transformed human endometrial stromal cells (T-HESCs) were incubated with medroxyprogesterone-17-acetate (MPA) and cyclic adenosine monophosphate (cAMP) for 4 days to induce decidualization. The expression levels of the decidualization markers prolactin (PRL) and insulin-like growth factor-binding protein-1 (IGFBP-1) were determined using quantitative polymerase chain reaction. RARα was knocked down using a small interfering RNA, and C/EBPβ was overexpressed from an adenoviral vector. The transcriptional regulation of CEBPB by RARα was determined by chromatin immunoprecipitation (ChIP) assay and luciferase assays.ResultsWe found that the expression levels of RARα decreased in the mid-luteal endometria of RIF patients. After 4 days of decidualization induction in vitro, RARα knockdown impaired the decidualization of T-HESCs and downregulated the expression of C/EBPβ. The restoration of C/EBPβ expression rescued the RARα knockdown-induced suppression of T-HESC decidualization. In ChIP analysis of lysates from decidualized T-HESCs, the CEBPB promoter region was enriched in chromatin fragments pulled down using an anti-RARα antibody. However, the relationship between CEBPB transcription and RARα expression levels was only observed when the decidualization of T-HESCs was induced by the addition of cAMP and MPA. To identify the binding site of RARα/retinoid X receptor α, we performed luciferase assays. Mutation of the predicted binding site in CEBPB (-2,009/-1,781) decreased the transcriptional activity of the reporter. To confirm this mechanism, the expression levels of C/EBPβ in the mid-luteal endometria of RIF patients were determined and found to decrease with decreased RARα expression levels.ConclusionA deficiency of RARα expression in the mid-luteal endometrium inhibits decidualization due to the downregulation of CEBPB transcription. This is a potential mechanism contributing to RIF.